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G-Biosciences  1-800-628-7730  1-314-991-6034  technical@GBiosciences.com
A Geno Technology, Inc. (USA) brand name
femto-ELISA-AP Kit
Enzyme-Linked Immunosorbent Assay for alkaline
phosphatase labeled antibodies
(Cat. #786-112)
think proteins! think G-Biosciences www.GBiosciences.com
INTRODUCTION ................................................................................................................. 3
ITEM(S) SUPPLIED (CAT. # 786-112) .................................................................................. 3
STORAGE............................................................................................................................ 3
ADDITIONAL ITEMS REQUIRED .......................................................................................... 3
PREPARATION BEFORE USE ............................................................................................... 4
PROTOCOL ......................................................................................................................... 4
IMPORTANT INFORMATION .......................................................................................... 4
1. APPLY ANTIGEN TO EACH WELL WITH SUITABLE COATING BUFFER.......................... 4
2. BLOCKING STEP .......................................................................................................... 4
3. PRIMARY ANTIBODY REACTION ................................................................................. 4
4. WASHING STEP 1 ....................................................................................................... 4
4. SECONDARY ANTIBODY REACTION ............................................................................ 4
5. WASHING STEP - 2 ..................................................................................................... 5
6. SUBSTRATE REACTION ............................................................................................... 5
RELATED PRODUCTS .......................................................................................................... 6
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INTRODUCTION
Enzyme-Linked Immunosorbent Assay (ELISA) is one of the most sensitive and powerful
techniques for detecting proteins, chemicals, and drugs (antigens) in biological samples,
including serum, blood and urine. The key to an ELISA is the interaction of a known
antibody with the antigen of interest, where either the antigen or antibody is
immobilized on an ELISA plate micro-well. For sensitive detection of antigen-antibody
complex, secondary antibody labeled with alkaline phosphatase (AP) is used. GBiosciences femto-ELISA-AP kit is supplied with an enhanced blocking agent (NAPBlocker), an improved, ultra sensitive, stable colorimetric alkaline phosphatase
substrate (pNPP; p-Nitrophenyl phosphate), and femto-TBST wash buffer. The kit
components are enough for performing 1,000 reactions as per the protocol.
ITEM(S) SUPPLIED (Cat. # 786-112)
Description
Size
femto-ELISA-AP Substrate (pNPP; p-Nitrophenyl phosphate), Cat # 786-113
1 x 100ml
NAP-Blocker (2X)
1 x 250ml
femto-TBST (10X), Cat # 786-161
2 x 250ml
STORAGE
The kit is shipped at ambient temperature. Upon arrival store the kit components at 4°C.
The femto-ELISA-AP Substrate is light sensitive and should be protected from direct
sunlight or UV sources.
ADDITIONAL ITEMS REQUIRED
Highest purity primary antibody, alkaline phosphatase (AP)-labeled secondary antibody,
coating/binding buffer, microwell plate designed for immunoassays, microplate reader,
multichannel pipettor etc.
NOTE: It is important that microwell plates specifically designed and formulated for
ELISA should be used (polystyrene tissue culture plates are not recommended as they
often produce erratic background).
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PREPARATION BEFORE USE
1. Allow all reagents to come to room temperature before use.
2. 10X femto-TBST Dilution: Dilute the appropriate volume of supplied 10X femtoTBST to 1X with DI Water.
3. NAP-Blocker Dilution: Before use, gently shake the supplied NAP-Blocker bottle to
mix it. Use aseptic techniques for handling NAP-Blocker. Dilute the appropriate
volume of supplied 2X NAP-Blocker 1:2 with 1X femto-TBST.
PROTOCOL
Important Information
I.
I.The experimental condition recommended in this protocol are adequate for most
applications, however, variables such as primary and secondary antibody
concentration, incubation time etc. can be modified or adjusted to meet individual
assay needs.
II.
II.Each of the protocol steps should be evaluated for establishing the optimum
conditions that yield maximum sensitivity.
1. Apply Antigen to each well with suitable Coating Buffer
Add 100µl Antigen, diluted in a suitable coating buffer [e.g. phosphate buffered saline or
50mM Sodium carbonate (pH9.6) with 20mM Tris-HCl (pH 8.5)] to the ELISA plate wells
and incubate at room temperature for 1 hour. After incubation, invert the plate to
empty and tap out residual liquid.
2. Blocking Step
Add 300µl of diluted [1X] NAP-Blocker to each well and incubate the plate for 15-30
minutes. After incubation, empty the NAP-blocker from the plate and gently tap out the
residual liquid.
3. Primary Antibody Reaction
Add 100µl specific primary antibody solution (diluted in 1X NAP-Blocker) to each well
and incubate for 1 hour at room temperature. After incubation, empty the plate
carefully and gently tap out the residual liquid.
4. Washing Step 1
Fill each reaction well with 1X femto-TBST (~350µl) and wait for 30 seconds then invert
the plate to empty. Gently tap out the residual liquid from each well. Repeat the above
washing steps 4-5 times.
4. Secondary Antibody Reaction
Add 100µl AP-labeled secondary antibody solution (appropriately diluted in 1X NAPBlocker) into each well and incubate for 1 hour at room temperature. After incubation,
empty the liquid from each well and gently tap out the residual liquid.
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5. Washing Step 2
Fill each reaction well with 1X femto-TBST (~350µl) and wait for 30 seconds then invert
the plate to empty. Tap out the residual liquid from each well. Repeat the washing steps
4femto-TBST in each well and wait for 5 minutes.
Tap out the residual wash from each well.
6. Substrate Reaction
After washing step –II, add 100µl femto-ELISA-AP Substrate into each well. A soluble
yellow color develops, which can be read at 405-410nm ranges, using femto-ELISA-AP
Substrate as blank.
Fore best results, sample absorbance values should be monitored and read before
absorbance values exceed 2.0 OD units. To reduce the intensity of the reaction color, it
is recommended to dilute the antibodies or the conjugates. However, dilution of femtoELISA-AP Substrate is not recommended.
In end point assays, the substrate reaction can be stopped, by adding 50µl of 3N Sodium
hydroxide (NaOH) carefully to the reaction wells.
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RELATED PRODUCTS
Download our Assay Development Handbook.
http://info2.gbiosciences.com/complete-assay-development-handbook
For other related products, visit our website at www.GBiosciences.com or contact us.
Last saved: 06/19/2014 SM
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